Skin fibrotic and cirrhotic diseases are characterized by excessive collagen synthesis. For example, keloids are cutaneous lesions composed of excess accumulation of extracellular matrix. The predominant matrix component that is increased is collagen. Several treatment modalities are available including intralesional injections of glucocorticosteroids, surgical excision, cryotherapy and compression (Kelly, A. P. Keloids, Dermatol. Clin. 6:413-424, (1988)). Intralesional injections of human interferon alpha-2b (Berman, B., and M. R. Duncan, "Short-term keloid treatment in vivo with human interferon alfa-2b results in a selective and persistent normalization of keloidal fibroblast collagen, glycosaminoglycan, and collagenase production in vitro" J. Am. Acad. Dermatol. 21:694-702 (1989)); and topical applications of retinoids, (Panabiere-Castaings, M. H., "Retinoic acid in the treatment of keloids" J Dermatol Surg. Oncol., 14:1275-1276 (1988); Daly, T. J., and W. L. Weston, "Retinoid effects on fibroblast proliferation and collagen synthesis in vitro and on fibrotic disease in vivo", J Am Acad Dermatol., 15:900-902 (1986)), have also been employed. In the majority of therapeutic modalities, variable success has been encountered. Therefore, other alternative therapies need to be developed.
Heparin and endothelial cell growth factor are capable of influencing a variety of biologic activities of many cell types (for example, Clowes A. W., and M. J. Karnovsky, "Suppression by heparin of smooth muscle cell proliferation in injured arteries", Nature 265:625-626 (1977); Casteliot, J. J. Jr., A. M. Kambe, D. E. Dobson, and B. M. Spiegelman, "Heparin potentiation of 3T3-adipocyte stimulated angiogenesis: mechanisms of action of endothelial cells", J Cell Physiol., 127:323-329 (1986); Ehrlich, H. P., W. K. Jung, D. E. Costa, and J. B. M. Rajaratnam, "Effects of heparin on vascularization of artificial skin grafts in rats", Exp. Molec Pathol., 48:244-251 (1988); Joseph-Silverstein, J. and D. B. Rifkin, "Endothelial cell growth factors", Semin Thromb. Hemost., 13:504-513 (1987)).
Heparin, a highly negatively-charged glycosaminoglycan, has been demonstrated to inhibit vascular smooth muscle cell proliferation both in vivo and in vitro (A. W. Clowes et al., supra). Heparin has also been found to promote angiogenesis (J. J. Casteliot, Jr. et al., supra); modulate the synthesis of collagen by chondrocytes (Brown, C. C. and G. Balian, "Effect of heparin on synthesis of short chain collagen by chondrocytes and smooth muscle cells", J. Cell Biol., 99:1688-1695 (1987)); and intestinal smooth muscle cells (Graham, M. T., D. E. Drucker, H. A. Perr, R. F. Diegelmann, H. P. Ehrlich, "Heparin modulates human intestinal smooth muscle cell proliferation, protein synthesis and lattice contraction", Gastroenterology 93:801-809 (1987)); and stimulate the synthesis of fibronectin and thrombospondin by human smooth muscle cells (Lyons-Giordano, B., H. Conaway, and N. A. Kefalides, "The effect of heparin on fibronectin and thrombospondin synthesis by human smooth muscle cells", Biochem. Biophys. Res. Commun., 148:1264-1269 (1987)).
Endothelial mitogens, such as endothelial cell growth factor (ECGF), are expressed by various cells and tissues. ECGF exhibits various biologic effects in cell culture and is mitogenic for a variety of cell types, that include endothelial cells (J. Joseph-Silverstein et al., supra). Furthermore, ECGF has a strong affinity for heparin (J. Joseph-Silverstein et al., Supra). Heparin prolongs the half-life of the growth factor and is, therefore, able to potentiate the biologic activities of ECGF (Damon, D. H., R. R. Lobb, P. A. D'Amore, and J. A. Wagner, "Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life", J. Cell Physiol.). For instance, the proliferative effect of ECGF on vascular endothelial cells is potentiated by heparin (Thornton, S. C., S. N. Mueller, and E. M. Levine, "Human endothelial cells: use of heparin in cloning and long-term serial cultivation", Science, 222:623-625 (1983)).
It is well known that heparin binds ECGF (Maciag, T., T. Mehlman, and R. Friesel, "Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain", Science, 225:932-935 (1984)) and potentiates the effect of ECGF, for instance, on the growth of human endothelial cells (S. C. Thornton et al., supra). Several workers have shown that heparin prolongs the biologic half-life of growth factors by protection from proteolytic degradation (B. Lyons-Giordano et al., supra), and acid and heat inactivation (Gospodarowicz, D. and J. Cheng, "Heparin protects basic and acidic fibroblast growth factor from inactivation", J Cell Physiol , 475-484 (1986)). In addition, heparin increases the affinity of ECGF for cell surface receptors by inducing conformational changes in the ECGF peptide (Schreiber, A. B., J. Kenney, J. Kowalsky, R. Freisel, T. Mehlman, and T. Maciag, "The interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition", Proc. Natl. Acad. Sci. USA, 82:6138-6142, (1985)).
Tan, E. M. L., E. Levine, T. Sorger, G. A. Unger, N. Hacobian, B. Planck, and R. V. Iozzo, "Heparin and endothelial cell growth factor modulate collagen proteoglycan production in human smooth muscle cells", Biochem. Biophys. Res. Commun., 163:84-92 (1989) disclose the effects of heparin and endothelial cell growth factor (ECGF) on extracellular matrix production in human iliac smooth muscle cells. The cells were grown in a medium supplemented with heparin and ECGF, medium supplemented with ECGF and unsupplemented medium. In the presence of heparin and ECGF, collagen production was inhibited 91-95% as compared to cultures incubated with ECGF alone or without both supplemental factors. In contrast, the production of proteoglycans was elevated 2.5 fold in the presence of heparin and ECGF. Enzymatic digestion of the proteoglycans indicated that both large and small molecular weight chondroitin sulfate proteoglycans were markedly elevated, while dermaton sulfate and heparin sulfate proteoglycans were increased to a lesser extent suggesting that a combination of heparin and ECGF elicits modulation of extracellular matrix production in smooth muscle cells, with divergent effects on collagen and proteoglycan synthesis. Furthermore, it was determined that heparin exerts a coordinate down-regulation of various matrix genes, that include the collagen, fibronectin and decorin genes, in smooth muscle cells.
U.S. Pat. No. 4,879,287 (Saliba) discloses a broad spectrum of medical applications for heparin and related molecules. It describes heparin's prevention of cell and tissue destruction and promotion of healing. The use of heparin for keloid prevention is taught. The invention comprises applying heparin/heparin-like compounds, either in solution or in the form of cream or aerosol, preferably at a pH of about 5.5, in an effective amount for a time sufficient to effect treatment. The concentration of heparin/heparin-like compounds is between 1500-5000 I.U./ml.
U.S. Pat. No. 4,745,098 (Michaeli) discloses compositions to improve wound healing comprising a suspension of collagen and glycosaminoglycan, such as heparin. Concentrations of collagen of 7-10 mg/ml in combination with 250-350 .mu.g/ml of the glycosaminoglycan are taught.
U.S. Pat. No. 4,837,024 (Michaeli) relates to compositions of fibrous protein, collagen, and a polysaccharide such as heparin which are applied with a bandage article to promote the healing of wounds.
U.S. Pat. No. 4,808,570 (Michaeli) discloses a suspension comprising fibrous protein, collagen, and glycosaminoglycan such as heparin used to improve the healing of wounds. The application promotes vascularization and attracts fibroblasts and endothelial cells by chemotaxis. The invention is particularly useful in treating persistent ulcerated wounds.
U.S. Pat. No. 4,760,131 (Sundsmo et al.) discloses a topical aqueous composition comprising fibrillar collagen, heparin and ungranulated platelets or platelet releasate which promotes collagen syntheses and wound healing.
In the majority of the prior art modalities for the treatment of skin fibrotic and other cirrhotic diseases, however, variable success has been encountered. Therefore, alternative efficacious therapies are still needed. An effective method of modulating collagen synthesis would therefore be useful therapeutically.